HCFs and HKCs were plated at a seeding density of 1 × 106 cells/well in 1.5 mL of RM on top of 6-well Transwell plates (Corning Life Sciences, Corning, NY). The following morning, all cells were stimulated at concentrations of 5 (LH) or 10 (FSH) mIU/mL in vitamin C media—Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 1% antibiotic/antimycotic, and stable vitamin C (0.5 mmol/L 2-O-α-D -glucopyranosyl-l -ascorbic acid, Sigma-Aldrich, St. Louis, MO)—as previously reported.29 (link), 30 (link), 31 (link), 32 (link) Constructs were stimulated with vitamin C media to stimulate matrix secretion and assembly. Fresh media were provided three times a week for the duration of the study (total of 4 weeks), as previously reported.29 (link), 30 (link), 31 (link), 32 (link)
2 o α d glucopyranosyl l ascorbic acid
Manufactured by Merck Group
Sourced in United States
2-O-α-D-glucopyranosyl-L-ascorbic acid is a chemical compound that is a derivative of L-ascorbic acid (vitamin C). It is a stable, water-soluble form of vitamin C.
Automatically generated - may contain errors
4 protocols using 2 o α d glucopyranosyl l ascorbic acid
1
Hormone-Induced Matrix Secretion and Assembly
2
3D Culture of HCFs and HKCs
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Male and female HCFs and HKCs were cultured in T175 flasks until ~80% confluency. Both cell types were counted following trypsinization and sub-cultured at a density of 1 × 106 cells/well in 6-well 3D (transwell) plates (VWR, Radnor, PA, USA) with 10% FBS, 1% AA in EMEM. Control constructs were stimulated with EMEM containing 10% FBS, 1% AA, and 0.5 mM stable vitamin C (0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid, Sigma-Aldrich, St. Louis, MO, USA), as previously reported [57 (link),58 (link),59 (link),60 (link)]. Constructs were also treated with E1 and E3 at three concentrations each as follows: E1—10 pg/mL, 50 pg/mL, and 150 pg/mL (E1253, Sigma-Aldrich, St. Louis, MO, USA) and E3—2 ng/mL, 15 ng/mL and 30 ng/mL (E9750, Sigma-Aldrich, St. Louis, MO, USA) in EMEM containing 10% FBS, 1% AA, and 0.5 mM stable vitamin C. All constructs were cultured for four weeks before further analysis.
3
3D and 2D Cell Culture Constructs
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Three-dimensional constructs - Stromal Cells: HCFs and HKCs were plated at a density of 1 × 106 cells/well on six-well size polycarbonate membrane inserts with 0.4-μm pores (VWR, Radnor, PA, USA). The cells were cultured in EMEM containing 10% FBS, 1% antibiotic, and stimulated with a stable Vitamin C derivative (0.5 mM 2-O-α-d -glucopyranosyl-l -ascorbic acid: Sigma-Aldrich, St. Louis, MO, USA). Cultures were grown for a total of 4 weeks and fresh media was supplied every other day for the duration of the study. Protein extraction and further analysis occurred at the 4-week timepoint.
Two-dimensional conventional cultures - Epithelial Cells: Cells were brought up from liquid nitrogen and cultured in a T75 flask in epithelial media, consisting of Keratinocyte Growth Media 2 PromoCell (VWR, Radnor, PA, USA), Penicillin-Streptomycin Solution (ThermoFisher, Rockford, IL, USA), and CaCl2 (Sigma-Aldrich, St. Louis, MO, USA). Once confluent, cells were removed via trypsinization and seeded in a 6-well plate at 1 × 106 cells/well in epithelial media. Cells were cultured at 37 °C for 24 h before protein was extracted [27 (link),28 (link)].
Two-dimensional conventional cultures - Epithelial Cells: Cells were brought up from liquid nitrogen and cultured in a T75 flask in epithelial media, consisting of Keratinocyte Growth Media 2 PromoCell (VWR, Radnor, PA, USA), Penicillin-Streptomycin Solution (ThermoFisher, Rockford, IL, USA), and CaCl2 (Sigma-Aldrich, St. Louis, MO, USA). Once confluent, cells were removed via trypsinization and seeded in a 6-well plate at 1 × 106 cells/well in epithelial media. Cells were cultured at 37 °C for 24 h before protein was extracted [27 (link),28 (link)].
4
Extracellular Matrix Secretion Protocol
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HCFs, T1DMs, and T2DMs were seeded at a density of 1 × 106/well on 0.4 μM polycarbonate membranes (Corning, Corning, NY, USA), as previously described [27 (link), 30 (link)–32 (link)]. Cultures were maintained in EMEM, 10% FBS, and 1% AA and further stimulated by 0.5 mM stable vitamin C (VitC; 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid, Sigma-Aldrich, St. Louis, MO, USA) in order to promote secretion and assembly of extracellular matrix (ECM). The constructs were maintained in VitC media for 2 or 4 weeks with media changes occurring every other day [29 (link)].
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